Techniques will be further developed for in situ selective alkylation of proteins which play a crucial role in respiratory control and coupled phosphorylation. Radioactive uncoupler molecules will be designed, synthesized and used to alkylate the proteins at the coupling sites of heart mitochondria. The labeled protein subunits will be separated by gel permeation and ion- exchange chromatography, electrophoresis and isoelectric focussing, and then have their amino acid sequences determined. Important coupling factors and respiratory electron carriers will be isolated by known procedures, treated with these labeling uncouplers and the resulting radioactive polypeptides sequenced in a similar way. By comparing the amino acid sequences of the radioactive polypeptide from each in situ labeled protein with the corresponding radioactive polypeptides obtained by labeling the previously isolated protein, we hope to identify these in situ labeled proteins and their functional groups. Attempts will also be made to determine the topographical relationship between these membrane proteins by using radioactive uncouplers with two labeling groups on each uncoupler molecule. It is almost certain that the information obtained in this way will be very useful for the eventual elucidation and control of this central energy transducing process which regulates growth and keeps us in the living state. In view of the well-known carcinostatic activity of alkylating agents and the recent rapid progress of immunological research related to cancer therapy, we also plan to apply the present technique to antibody-directed in situ alkylation of specific proteins. Hopefully the methodology so developed will also be useful to workers in applied medical research, particularly in cancer chemotherapy.